Antineoplastic agents. 595. structural modifications of betulin and the X-ray crystal structure of an unusual betulin amine dimer1

Article abstract of DOI:10.1021/np400947d

The lupane-type triterpene betulin (1) has been subjected to a series of structural modifications for the purpose of evaluating resultant cancer cell growth inhibitory activity. The reaction sequence 7 11 12 was especially noteworthy in providing a betulin-derived amine dimer. Other unexpected synthetic results included the 11 and 13/1417 conversions, which yielded an imidazo derivative. X-ray crystal structures of dimer 12 and intermediate 25 are reported. All of the betulin modifications were examined for anticancer activity against the P388 murine and human cell lines. Significant cancer cell growth inhibition was found for 4, 8, 9, 15/16, 19, 20, 24, and 26, which further defines the utility of the betulin scaffold.

Full text of DOI:10.1021/np400947d

Article
pubs.acs.org/jnp
Antineoplastic Agents. 595. Structural Modifications of Betulin and
1
the X‑ray Crystal Structure of an Unusual Betulin Amine Dimer
George R. Pettit,* Noeleen Melody, Frank Hempenstall, Jean-Charles Chapuis, Thomas L. Groy,
and Lee Williams‡
Department of Chemistry and Biochemistry, Arizona State University, P.O. Box 871604, Tempe, Arizona 85287-1604, United States
*
S Supporting Information
ABSTRACT: The lupane-type triterpene betulin (1) has been sub-
jected to a series of structural modifications for the purpose of eval-
uating resultant cancer cell growth inhibitory activity. The reaction
sequence 7 → 11 → 12 was especially noteworthy in providing a betulin-
derived amine dimer. Other unexpected synthetic results included the 11
and 13/14 → 17 conversions, which yielded an imidazo derivative. X-ray
crystal structures of dimer 12 and intermediate 25 are reported. All of the
betulin modifications were examined for anticancer activity against the
P388 murine and human cell lines. Significant cancer cell growth inhibition
was found for 4, 8, 9, 15/16, 19, 20, 24, and 26, which further defines the
utility of the betulin scaffold.
xtracts of birch bark (Betula species) usually contain a
mixture of betulin (1) as a major component accompanied
NCI in September 1957 to advance a carefully targeted research
E
program (CCNSC) for the discovery and development of
important, new naturally occurring and synthetic drugs for
improving human cancer treatments. That same month was the
start of our group’s immediate commitment to the discovery of
new anticancer drugs derived from natural products and the
beginning of our continuing collaboration with the NCI. The first
such collaborative research was aimed at evaluating species of the
Labiatae family, one of several known at the time to be most
by related pentacyclic triterpenoids and have been used in
2a
traditional medicine in local areas through the ages. Although
betulin (1) has only moderate anticancer, antibacterial,
antifungal, and antiviral activity and other biological properties,
structural modifications are readily accessed to provide
derivatives both known and new that have improved potency
2
−4
in these areas.
Betulin (1) is a relatively accessible starting
2
6
molecule for such medicinal chemistry research. Among other
avenues, we have employed betulin in SAR synthesis efforts
focused on cephalostatin 1 (2), a powerful marine organism
frequently used in traditional cancer treatments.
In that early period we were also evaluating other plant
constituents such as those from the white birch (Betula
papyrifera) bark. We pursued the lupanes (A), the betulin/
5a−c
anticancer constituent.
7
betulinic acid series from birch bark, and the 11-ene oleanane
(
B)/oleanolic acid and the 11-ene ursole (C)/ursolic acid from
6
salvia species. However significant anticancer activity was not
detected using the then current NCI evaluation system
(
generally murine in vivo Walker 256 sarcoma and/or leukemia
L1210). Subsequently a large number of advances in both
2
triterpenoid chemistry and cancer cell growth inhibition
evaluations have shown the above early triterpene leads to
have cancer cell growth inhibitory activity. Indeed triterpenes
2
,3
representing type A (including betulinic acid) are now in
4
8
9
human cancer clinical trials, and B and C have generated
considerable scientific and medicinal interest due to the in-
creasing diverse types of biological activities, which now include
10
antiviral.
Cephalostatin 1 (2) was first isolated by our group in 1988
5a−c
from the Indian Ocean marine worm Cephalodiscus gilchristi.
The natural product is a unique bis-steroid, biosynthetically
Nature’s infinite ability in biosynthesis productivity and the
promise of the discovery of new anticancer drugs inspired the
Received: November 15, 2013
Published: April 2, 2014
©
2014 American Chemical Society and
American Society of Pharmacognosy
863
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Journal of Natural Products
Article
constructed by the organism in an unsymmetrical coupling of
two C steroidal units by way of the C-2/C-3 pyrazine ring. The
Scheme 1
27
11
first total synthesis of this molecule was achieved in 1998. In
order to advance our ongoing structure−activity relationship
investigations of this promising anticancer lead, we attempted the
synthesis of pyrazino-bis-lupane 3 in order to further explore
structure−activity boundaries.
RESULTS AND DISCUSSION
■
The first approach pictured a reductive dimerization of 2-azido
ketone 10. However the synthesis and isolation of azide 10
proved difficult, as it decomposed rapidly to the enamine 11,
which, in turn, provided the novel betulin dimer 12 (Scheme 1).
The dimer was crystallized, and the structure elucidated using
X-ray crystallography (Figure 1). Both the dimer and its pre-
cursors were evaluated against murine and human cancer cell
lines (Table 1). The absence of significant cancer cell growth
inhibition with 12 caused by the presumed rotation around the
secondary amine bridge of dimer 12 suggests a critical need for a
strictly rigid pyrazine skeleton. However, our and others’
experience probing for a route to structurally simple steroid
pyrazine dimers based on the cephalostatins indicates a need for
more closely approximating the cephalostatin E and F ring
an orange resin, which contained several dimers, detected by
mass spectroscopic analysis. The 2-azido ketone 10 was not
isolated. If the reaction was carried out at room temperature (rt)
with 1.3 equivalents of NaN , then a 2α/β-azido ketone mixture
3
was isolated but decomposed in air to a red solid mixture. When
the 2-bromo ketone was treated with NaN on a small scale
3
(30 mg) in either DMF or N-methyl-2-pyrrolidine (NMP) using
15a−c
a catalytic amount of HOAc,
the 2α/β-azido ketone mix-
1
2
complexity.
Acetylation of the primary hydroxy group of 1 yielded the
ture 10 was obtained along with a minor amount of enamine
11. Upon scale-up, 11 was again the major product and de-
composition of the material to a red solid mixture was observed.
13
monoacetate 28-O-acetylbetulin 4 as the major product (42%)
and the diacetate 5 as a minor component (15%). The com-
pounds were separated using silica gel column chromatography
11
When tetramethylguanidinium azide (TMGA) in acetoni-
trile was used instead of NaN , the result was a mixture of
3
14
(
SGC, DCM). Oxidation of 4 using pyridinium dichromate
the α-azido ketone (10), the enamine (11), and the starting
bromide.
14
gave the ketone 6 in good yield (92%). Bromination of 6 using
phenyltrimethylammonium tribromide yielded a mixture of 2α-
and 2β-bromo ketones 7/8 (57/31%), which was separated by
SGC. Upon scale-up, the dibromo derivative 9 was also isolated
Owing to the rapid decomposition of the azido ketone 10 to
the enamine 11, reaction products (obtained from the reaction
of 7 with TMGA/DMF/CH CN or with NaN /DMF) were
3
3
(
15%). The dibromo derivative was unstable upon standing and
immediately allowed to react without further purification using
16
decomposed over time.
the Staudinger reaction with Ph P in the hope of forming the
3
Treatment of the bromo ketone (7/8) mixture with excess
iminophosphorane from any azide remaining in the product
mixtures. The phosphorane would then be hydrolyzed in
aqueous THF with the prospect of obtaining the 2-amino
ketone, which was expected to spontaneously dimerize and
NaN in DMF and a catalytic amount of NaI afforded the
3
14
azide 10, which rapidly decomposed to the enamine 11. The
enamine was also unstable and decomposed over some time to
8
64
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Journal of Natural Products
Article
reaction products were subjected directly to the Staudinger
reaction. After air oxidation in EtOH with a catalytic amount of
p-toluenesulfonic acid, a bright yellow, amorphous powder pre-
cipitated following several days of stirring at rt. The precipitate
was recrystallized from DCM/MeOH to yield crystals of 12,
whose structure was confirmed by X-ray crystallography. This
product was not obtained when enamine 11 was stirred in EtOH
and a catalytic amount of p-toluenesulfonic acid for several days
at room temperature.
Hydrogenation of the azido/enamine mixtures was also
performed using 10% Pd/C in a mixture of MeOH and EtOAc
19
containing a catalytic amount of glacial HOAc. Again, the
reaction did not yield pyrazine 3. In the event that amine prod-
ucts were present in this unresolved mixture, a catalytic amount
of p-toluenesulfonic acid in EtOH was employed to promote
dimerization, but with no effect.
The route to the pyrazine via the unstable azide 10 was un-
successful, and a further attempt at obtaining 3 using a different
approach involved condensation of 11 with 2-hydroxy ketone 13
20a,b
was pursued as outlined in Scheme 2.
The preparation and
purification of 2α-hydroxy ketone 13 again presented difficulties
1
8
and was obtained as the major isomer accompanied by the 2-oxo-
Figure 1. X-ray crystal structure of dimer 12. Hydrogen atoms and
3
-hydroxy derivative 14. The α-orientation of the C-2 hydroxy
labels have been omitted for clarity. Thermal ellipsoids are shown at the
=
50% probability level.
group of 13 was assigned based on the coupling constants J1α,2β
1
1
3 Hz and J1β,2β = 6 Hz. SGC [DCM/acetone (1.5%)] of the
3/14 regiomeric mixture gave an inseparable mixture in a ratio
undergo autoxidation to give pyrazine 3, as is routinely observed
for α-amino ketones under these conditions.
No pyrazines were isolated using this method from either
reaction mixture. When the experiment was carried out with
of 50:50. Further attempts at purification of this mixture by
chromatography on silica gel eluting with hexanes/EtOAc (4:1)
gave a compound that was slow to elute and was found to be
the 2-oxo-3-hydroxy-28-O-acetylbetulin derivative 14. The 2-
hydroxy ketone 13 was not isolated and may have decomposed
15c,17
epimers 7/8 using the NaN /DMF/NaI method, again the
3
Table 1. Murine and Human Cancer Cell Line Data [ED and GI , μg/mL]
5
0
50
a
cell line
SF-268
compound no.
P388
BXPC-3
MCF-7
NCI-H460
KM20L2
DU-145
1
4
5
6
7
8
9
9.3
4.1
>10
3.0
>10
4.0
7.4
2.5
>10
5.0
>10
7.6
0.805
0.806
0.12
>10
>10
3.1
>10
8.0
>10
10.6
10.1
3.4
>10
5.2
>10
12.7
9.6
>10
>10
9.0
0.725
4.1
12.8
2.8
>10
0.391
3.4
3.2
4.4
6.1
1.0
2.5
4.4
4.4
4.2
3.8
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
0
6.6
5.0
>10
>10
>10
7.0
4.4
4.2
5.7
1
>10
7.9
>10
>10
4.8
>10
>10
6.2
>10
>10
5.1
>10
>10
8.8
2
>10
>10
>10
>10
>10
5.6
3/14
4
9.1
5.8
6.1
8.0
5.9
>10
9.4
5/16
7
3.4
3.8
4.2
3.2
4.0
14.5
1.9
11.2
2.0
>10
6.9
>10
2.8
>10
4.9
>10
5.8
7a
8
>10
3.1
>10
5.0
>10
3.4
>10
2.9
>10
2.7
>10
6.7
9
0
3.1
4.7
8.7
3.8
7.9
4.5
1
>10
10.8
>10
2.4
7.1
>10
>10
>10
2.7
8.8
8.0
9.3
2
11.2
>10
2.7
>10
>10
2.3
>10
>10
2.2
>10
>10
2.3
3
4
>10
>10
5
8.2
11.6
6.1
>10
>10
13.1
4.4
>10
>10
>10
>10
6
3.9
a
Cancer cell lines in order: murine lymphocytic leukemia (P388); lung (NCI-H460); colon (KM20L2); prostate (DU-145); pancreas (BXPC-3);
breast (MCF-7); CNS (SF-268).
8
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Scheme 2
Scheme 3
on the column. Acetylation of the hydroxy ketone (13/14)
mixture in order to simplify separation gave a mixture of
acetylated products 15/16. That served to confirm the 2-
hydroxy/3-hydroxy ketone mixture as the starting material and
was further established by deshielded H-2 and H-3 resonances
due to the C-2 and C-3 acetoxy groups, with the coupling con-
stants for the H-2β resonance [J1α,2β = 13 Hz and J1β,2β = 6 Hz] of
in low yield following a six-day stirring period. The solid
precipitate showed an MS ion at m/z = 890 corresponding to
dimer 23, presumed to be the allobetulin analogue of 12, and was
submitted for biological evaluation. The dimer 23 was also
obtained in low yield when the enamine 22 was stirred for 2 days
in EtOH with a catalytic amount of p-toluenesulfonic acid.
Hydrogenation of the enamine (22) over 10% Pd/C gave a polar
mixture of products that resisted separation.
1
5 augmented by the H-3 singlet in compound 16. The expected
1
absence of the two hydroxy resonances in the H NMR spectrum
and the presence of two O-acetyl resonances at δ 2.18 and 2.14
were observed.
The enamine (11) was then allowed to react with the hydroxy
ketone mixture 13/14 in MeOH/DCM in the presence of
In a continued effort to moderate possible steric effects
presented by the C-4 dimethyl group, the synthesis of the 2-oxo-
methyl ester 25 was performed (Scheme 4) using the
NH OAc employing controlled addition of the hydroxy ketone
4
22
mixture to prevent its homodimerization. The products were
separated by SGC to yield another mixture of dimers (by MS
analysis) that resisted separation, along with compound 17,
whose structure was proposed based on 1D NMR (APT) and 2D
NMR (HSQC, COSY, HMBC) analysis. Deprotection of the
C-28 acetate group with K CO gave the alcohol with MS and
Willgerodt−Kindler reaction and was isolated along with the
23
2-morpholino-3-oxo derivative 26 from ketone 24. The struc-
ture of 25 was elucidated using X-ray crystallography on crystals
obtained by recrystallization from MeOH (Figure 2). Bromina-
tion of 25 yielded a mixture of brominated products, and
treatment with NaN followed by azide reduction with PPh in
2
3
3
3
NMR data supporting structure 17a. Clearly, the considerable
steric constraints caused by the C-4 gem-dimethyl group in a 1,3
relationship with the C-10 methyl group imposed a series of
synthesis challenges.
Finally, in order to eliminate any unpredicted steric and/or
unwanted chemical interactions from the betulin E-ring olefin and
THF gave complex mixtures. Preliminary evaluation of such
mixtures using MS data and NMR analysis indicated that this
route did not yield any pyrazines and the synthetic methods
utilized lacked the necessary practicality to be refined.
All compounds prepared in the study were examined for
cancer cell growth inhibition (Table 1) against a series of murine
and human cancer cell lines. The cancer cell growth inhibition
of betulin improves upon acetylation at C-28 (4). However
diacetate 5 was considered inactive. When the monoacetate 4 was
oxidized to the C-3 oxo derivative (6), a decrease in activity against
some cell lines resulted. Bromination at C-2 (7/8) caused a return
to the modest cancer cell growth activity seen for the parent
molecule. The β-bromo isomer gave slightly higher activity when
compared to the α-isomer. Bromination at C-2 was previously
2
2
8-OAc functionalities, preparation of 2-azido-3-oxoallobetulin
0 was undertaken from 2α-bromo-3-oxoallobetulin 19 using
21
published methods (Scheme 3). However this method gave
inconsistent results. A mixture containing only enamino ketones
was obtained in a repeat experiment and used in the azide reduc-
tion procedure with triphenylphosphine. Chromatographic
separation of the products did not yield dimers, and the products
were identified as the enolic ketone 21 and the enamine 22.
When the products from the preparation of 20 were not
separated by column chromatography but taken directly to the
reduction step followed by air oxidation in EtOH with a catalytic
amount of p-toluenesulfonic acid, an amorphous powder precipitated
24
shown to improve the activity of certain triterpenoid derivatives.
The enamine 10 and the dimer 12 proved to be inactive.
In summary, attempts at the synthesis of a bis-pyrazine-lupane
using betulin, instead, led to a new lupane dimer (12). The 2-azido
8
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Sigma-Aldrich Chemical Co. and were used as received. For thin-layer
chromatography, Analtech silica gel GHLF Uniplates were used
and visualized with short-wave UV irradiation and use of an iodine
chamber or a 10% H SO /EtOH dip followed by heating. Solvent
Scheme 4
2
4
extracts of aqueous solutions were dried over MgSO . For column
4
chromatography, silica gel (230−400 mesh ASTM) from E. Merck
(Darmstadt, Germany) was used. Melting points are uncorrected and
were determined with a Fisher−Johns melting point apparatus. Optical
rotations were measured by use of a Perkin-Elmer 241 polarimeter, and
−1
2
−1
1
13
the [α] values are given in 10 deg cm g . H and C NMR spectra
D
were recorded on Varian Unity INOVA 400 and 500 instruments with
deuterated solvents. HRMS were obtained with a Jeol JMS-LCmate
mass spectrometer. Elemental analyses were determined by Galbraith
Laboratories, Inc. The X-ray crystal structure data were obtained on a
Bruker APEX2 CCD diffractometer using Mo Kα (0.71073 Å) radiation.
Betulin (1). Small quantities of betulin (1) were purchased from
2
5
Sigma-Aldrich, and larger quantities were isolated from the bark of
birch trees (Betula papyrifera), collected in the state of Maine by
extraction with hot toluene (80 °C) for 3 h. The crude extract was
washed with aqueous 5% K CO , dried (MgSO ), and recrystallized
2
3
4
from hot toluene to yield 1 as a colorless solid: R = 0.11 (DCM); mp
f
1
3
1
13
2
52−255 °C; lit. mp 250−252 °C; H and C NMR data were
26a,b
consistent with those reported.
2
8-O-Acetylbetulin (4) and 3,28-Di-O-acetylbetulin (5). A solution
of betulin (1) (1.0 g, 2.3 mmol), Ac O (12 mL, 0.124 mol, excess), and
2
imidazole (0.35 g, 4.6 mmol) in CHCl (40 mL) was heated under reflux
3
(
61−62 °C) until TLC (DCM) showed product development was
almost complete (∼2 h). The reaction mixture was cooled to room
temperature and washed with 10% HCl, (10 mL), H O (10 mL), and
2
brine (10 mL) and dried (Na SO ), and the solvent was removed
2
4
under reduced pressure. TLC indicated the presence of three com-
pounds. Separation was achieved using SGC with DCM as the eluent
to give 3,28-di-O-acetylbetulin 5 (0.16 g, 15%): R 0.5 (DCM); mp
f
1
3
20
13
2
[
28−229 °C, lit. mp 216−218 °C; [α] +15 (c 0.4, CHCl ), lit.
D
3
20 1 13
α] +20 (c 1.67, CHCl ); H and C NMR spectroscopic data of 5
D 3
26a,c
were in agreement with published data;
HREIMS m/z 526.3999
(
calcd for C H O , 526.4022); 28-O-acetylbetulin (4) (0.454g, 42%);
34
54
4
1
3
20
R 0.2 (DCM); mp 210−212 °C, lit. mp 210−212 °C; [α]
(
spectroscopic data of 4 were in agreement with published data;
+6
D
f
1
3
20
1
13
c 0.4, CHCl ), lit. [α] +8.5 (c 1.58, CHCl ); H and C NMR
3 D 3
2
6c 13
C
NMR (CDCl , 100 MHz) δ 171.6, 150.1, 109.8, 78.9, 62.8, 55.3, 50.3,
3
4
2
8.7, 47.7, 46.3, 42.7, 40.8, 38.8, 38.7, 37.5, 37.1, 34.5, 34.2, 29.7, 29.5,
7.9, 27.4, 27.0, 25.2, 21.0, 20.8, 19.1, 18.3, 16.1, 16.0, 15.4, 14.7, and
betulin (1) (0.117 g, 11%).
-Oxo-28-O-acetylbetulin (6). The monoacetate 4 (0.75 g, 1.55
3
mmol) was dissolved in dry DCM (40 mL) and stirred under N at room
2
1
8
temperature. Pyridinium dichromate (0.77g, 1.99 mmol) was added.
The mixture was a bright orange color initially, which darkened over
time. The reaction was allowed to proceed for a total of 20 h, and at this
point starting material was present as a faint spot by TLC (DCM). The
Figure 2. X-ray crystal structure of ketone 25. Hydrogen atoms and
labels have been omitted for clarity. Thermal ellipsoids are shown at the
50% probability level.
mixture was filtered through silica gel and eluted with Et O (500 mL).
2
The Et O layer was concentrated to a colorless foam solid (0.73 g, 98%).
ketone 10 was difficult to prepare and was obtained as part of a
mixture containing the slightly more stable enamino ketone 11 or
2
Further purification by SGC (eluent: DCM) gave the ketone 6 as a
2
7a
colorless solid: mp 81−82 °C (0.69 g, 92%); R 0.41 (DCM); lit. mp
f
22. A series of methods aimed at dimerization and hence pyrazine
20
27a
20
1
17−119 °C; [α] +29 (c 0.2, CHCl ), lit. [α] +38 (c 1.35,
CHCl ); H and C NMR spectroscopic data of 6 were in agreement
with published data; HREIMS m/z 482.3779 (calcd for C H O ,
D
3
D
formation of a 10/11 mixture failed to yield the target pyrazines.
The dimer 12 was the only pure compound isolated from the
reaction product mixtures when 10/11 was allowed to react in an
aza-Wittig-type reaction followed by air oxidation. That suggests
the bulky C-4 gem-dimethyl group and the 1,3 steric relationship
with the axial C-10 methyl group interfered with the formation of a
pyrazine dimer. Similar compounds lacking the C-4 gem-dimethyl
group readily dimerize under these conditions to give pyrazines.
However, the specialized chemical considerations learned here will
be useful to our future research in this field.
1
13
3
27b
3
2
50
3
4
82.3760); anal. C 74.04, H 10.32%, calcd for C H O , C 79.62, H
32 50 3
10.44%.
2α/β-Bromo-3-oxo-28-O-acetylbetulin 7/8 and 2,2-Dibromo-3-
oxo-28-O-acetylbetulin 9. A solution of 6 (0.10g, 0.2 mmol) in
anhydrous THF (5 mL) was stirred at 0 °C under N . Phenyl-
2
trimethylammonium tribromide (0.085 g, 0.25 mmol, 1.3 equiv) was
dissolved in THF (5 mL) and cooled to 0 °C before adding to the
solution of the starting material in THF. The mixture was stirred for 2 h,
quenched with brine, and diluted with EtOAc. The organic layer was
separated and washed with brine followed by H O, dried (Na SO ),
2
2
4
EXPERIMENTAL SECTION
General Experimental Procedures. Reagents and anhydrous
solvents were purchased from Acros Organics (Fisher Scientific) and
■
filtered, and concentrated to a colorless oil. TLC showed two products
when compared to the starting material in DCM, and these were
1
separated by flash SGC (50 g, column size 40 × 1 / cm, elution rate:
2
8
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Journal of Natural Products
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1
mL/0.5 min). 2α-Bromo-3-oxo-28-O-acetylbetulin (7) (0.066g, 57%):
1,2, 1′,2′-Ene, 2,2′-Bisamino-3,3′-oxo-28,28′-O-acetylbetulin 12.
To a solution of 2α/β-bromo-3-oxo-28-O-acetylbetulin (7/8) (0.68 g,
1.2 mmol) in anhydrous DMF (40 mL) under N were added NaN
1
mp 114 °C; R (DCM) 0.38; H NMR (CDCl , 400 MHz) δ 5.05 (1H,
dd, J = 13.4, 6.2 Hz, H-2), 4.67, 4.58 (each 1H, nm, H-29), 4.24, 3.82
f
3
2
3
(
(
1
each 1H, d, J = 12 Hz, H-28), 2.62 (1H, dd, J = 12.8, 6.0 Hz, H-2 e), 2.44
(1.0 g, 15.6 mmol, 13 equiv) and a catalytic amount of NaI. The mixture
was stirred at 50 °C under nitrogen for 2 h. The solvent was evaporated
under reduced pressure, keeping the solution at 43−45 °C using an oil
1H, ddd, J = 11, 5.5 Hz, H-19), 2.07 (3H, s, OCOCH ), 1.66, 1.17, 1.11,
3
13
.07, 1.06, 0.95 (each 3H, s, CH ), 1.97−1.00 (CH, CH ); C NMR
3
2
(
CDCl , 100 MHz) δ 207.0, 171.6, 149.9, 110.1, 62.7, 56.6, 52.7, 52.6,
bath. When the mixture was completely dry, CHCl (50 mL) was added
3
3
4
2
5
9.7, 49.3, 48.6, 47.6, 46.2, 42.8, 41.0, 39.9, 37.5, 34.5, 33.7, 29.6, 29.5,
7.0, 26.2, 24.9, 21.7, 21.0, 19.2, 19.1, 16.0, 15.9, 14.6; HREIMS m/z
60.2889 (calcd for C H BrO , 560.2865); anal. C 67.48, H 8.70%,
and the organic solution was washed with brine (2 × 20 mL), dried
(Na SO ), and concentrated to yield 11, which was stored under
2
4
vacuum and taken directly to the next step. To a solution of enamine 11
32
49
3
calcd for C H BrO , C 68.43, H 8.79 %.
in anhydrous THF (20 mL) was added Ph P (1.0 g, 3.81 mmol, 3 equiv).
32
49
3
3
2
β-Bromo-3-oxo-28-O-acetylbetulin (8) (0.036 g, 31%): R (DCM)
The yellow-colored solution was stirred under N at rt for 24 h, then
heated at 40 °C for 22 h. Water (0.5 mL) was added, and the reaction
mixture was stirred at rt for 24 h, then concentrated and dried on a
f
2
1
0
4
2
.28; H NMR (400 MHz, CDCl ) δ 5.02 (1H, t, J = 10.2 Hz, H-2), 4.61,
.52 (each 1H, s, H-29), 4.17, 3.77 (each 1H, d, J = 11 Hz, H-28), 2.34−
.42 (2H, m, 2H, H-1e, H-19), 2.00(3H, s,OCOCH ), 1.61 (3H, s,
3
vacuum pump to remove H O. The yellow oil was taken up in EtOH
3
2
CH ), 1.05 (6H, s, 2 × CH ), 0.95 (6H, s, 2 × CH ), 0.89−1.99 (CH,
(20 mL), and an immediate precipitate was observed upon stirring at rt.
p-Toluenesulfonic acid was added (cat. amount), and the yellow mixture
was stirred at rt for 3 days and monitored by TLC. The reaction mixture
was concentrated to minimum volume, and a fine precipitate developed,
3
3
3
13
CH ), 0.72 (3H, s, CH ); C NMR (CDCl , 100 MHz) δ 209.0, 171.5,
2
3
3
1
4
1
49.8, 110.00, 62.6, 54.0, 52.0, 51.4, 49.6, 48.6, 47.5, 47.4, 46.2, 42.7,
0.8, 39.2, 37.8, 34.5, 32.6, 29.6, 29.5, 29.2, 26.9, 25.1, 21.9, 21.0, 20.0,
9.8, 19.1, 18.8, 15.3, 14.6. On scale-up the 2,2-dibromo derivative 9 was
which was filtered through Celite and washed with CH Cl and EtOAc.
3
also isolated (19% yield): mp 112−115 °C; R (DCM) 0.43; HREIMS
The combined washings and mother liquor were concentrated and
purified (gradient elution; hexanes/EtOAc, 9:1 → EtOAc 100%) to give
the main fraction as a yellow powder (0.153 g, 27%). Two successive
recrystallizations from DCM/MeOH afforded crystals of 12 suitable for
f
1
m/z 638.1945 (calcd for C H Br O , 638.1971); H NMR (CDCl
32
48
2
3
3,
4
00 MHz) δ 4.69, 4.60 (each 1H, s, H-29), 4.25, 3.85 (each 1H, d, J = 8
Hz, H-28), 3.62, 3.11 (each 1H, d, J = 16 Hz, H-1), 2.47 (1H, m, H-19),
20
1
2
1
.08 (3H, s, OCOCH ), 1.69 (3H, s, CH ), 1.23 (6H, s, 2 × CH ), 1.05,
X-ray crystallography: mp 205 °C, [α] −39 (c 0.2, CHCl ); H NMR
3
3
3
D
3
.01, 0.92 (each 3H, s, CH ); HREIMS m/z 638.1945 (calcd for
(CDCl , 400 MHz) δ 6.37 (3H, m, H-1, H-1′, N−H), 4.70, 4.62 (each
3
3
C H Br O , 638.1971).
2H, s, H-29, H-29′), 4.27, 3.83 (each 2H, d, J = 12 Hz, H-28, H-28′),
32
48
2
3
2
α/β-Azido-3-oxo-28-O-acetylbetulin, 10. To a solution of the
2.43 (2H, ddd, J = 10, 6 Hz, H-19, H-19′), 2.06 (6H, s, 2 × OCOCH ),
3
2
α/β-bromo ketone (7/8) mixture (0.03 g, 0.05 mmol) in DMF (1 mL)
2.0−1.03 (CH, CH ), 1.69 (6H, s, 2 × CH ), 1.14 (6H, s, 2 × CH ), 1.09
2
3
3
13
were added HOAc (0.01 mL) and NaN (0.02 g, 0.31 mmol, 6.2 equiv).
(18 H, s, 6 × CH ), 0.96 (6H, s, 2 × CH ). C NMR (CDCl , 100
3
3 3 3
The reaction mixture was stirred at rt for 1 h. Iced water was added, and
the precipitate was collected by filtration and dried under vacuum to
afford 20 mg (74% yield) of the α/β isomers with a minor amount of 11
as an impurity. Column chromatography was carried out using a
gradient elution of hexanes/EtOAc (6:1), affording the 2α/β-azido-3-
MHz) δ 200.8, 171.7, 149.9, 133.6, 132.4, 110.0, 62.8, 53.0, 48.7, 47.5,
46.38, 46.0, 44.4, 43.1, 41.7, 38.3, 37.8, 34.6, 33.9, 29.8, 29.7, 27.9, 27.0,
25.3, 21.8, 21.3, 21.1, 20.6, 19.4, 18.9, 16.5, 14.8; HRMS (APCI+) m/z
+
974.7530 [M + H ] (calcd for C H NO , 974.7232); anal. C 72.66, H
64
96
6
9.23, N 1.42%, calcd for C H NO ·CH Cl , C 73.69; H 9.23, N, 1.32%.
64
95
6
2
2
oxo-28-O-acetylbetulin mixture (10) as a colorless oil, which solidified
This compound was stored in the dark, as it is light sensitive and goes
from bright yellow to dark orange with prolonged exposure to light.
X-ray Crystal Structure of 12. a. Data Collection. A clear, pale
yellow, plate-like specimen (0.08 mm × 0.32 mm × 0.36 mm) of 12
grown from a DCM/MeOH solution was mounted on the end of a thin
glass fiber using Apiezon type N grease and optically centered. Cell
parameter measurements and data collection were performed at 123 ± 1 K
with a Bruker APEX2 CCD diffractometer using Mo Kα (0.71073 Å)
radiation. A sphere of reciprocal space was collected using the Multi-Run
1
on standing (8 mg, 28% yield): H NMR (CDCl , 400 Hz) δ 4.67, 4.57
3
(
(
(
each 2H, nm, H-29), 4.19−4.26 (4H, m, H-28a, H-2α, H-2β), 3.82
2H, m, H-28b), 2.42(1H, m, H-19), 2.27 (1H, dd, J = 12, 6 Hz,), 2.06
6H, s, OCOCH ), 1.67, 166, 1.124, 1.12, 1.11, 1.08, 1.07, 1.01, 1.00,
3
+
0
.95 (s, 12 CH ); HRMS (APCI) + m/z 524.3856 [M + H] (calcd for
3
C H N O , 524.3852).
32
50
3
3
The bulk of the product mixture decomposed on the column as a red
band appearing upon loading the material. The remaining fractions
contained mixtures (7 mg) and 2,3-dioxo-28-O-acetylbetulin (5 mg,
2
8a
scheme available in the Bruker APEX2 data collection package. Three
sets of frames were obtained, with each set using 0.50°/frame steps to
cover 182° in ω at fixed φ values of 0°, 120°, and 240°, respectively, for a
total of 1092 frames. Data were integrated and reduced using Bruker
6
%). Some of the baseline material was removed with DCM/MeOH 1%
to yield a yellow residue, 15 mg.
-Enamino-3-oxo-28-O-acetylbetulin (11). To a solution of the α/β
2
2
8b
isomers bromo-3-oxo-28-O-acetylbetulin (7/8) (0.6 g, 1.18 mmol) in
SAINT, resulting in 99.9% coverage of all unique reflections to a
anhydrous DMF (60 mL) under N were added NaN (0.84g, 13 mmol,
resolution of 0.830 Å with an average redundancy of 2.27.
2
3
11 equiv) and a catalytic amount of NaI. The suspension was heated
b. Crystal Data. C H NO , fw = 976.43, monoclinic, P2 , a =
64
97
6
1
to 50 °C for 1 h. The reaction was terminated by the addition of H O
13.5315(6) Å, b = 16.3954(7) Å, c = 14.1737(6) Å, β = 106.5240(10)°,
2
3
−3
−1
(
100 mL). The aqueous fraction was extracted with EtOAc/Et O (1:2,
V = 3014.6(2) Å , Z = 2, ρ = 1.076 Mg m , μ(Mo Kα) = 0.067 mm ,
2
c
2
× 200 mL). The combined organic extract was washed with H O and
λ = 0.710 73 Å, F(000) = 1072.
2
brine (Na SO ) and concentrated under vacuum to yield a yellow solid
c. Structure Solution and Refinement. A total of 25 001 reflections
were measured, of which 11 004 were independent (Rint = 0.0209) and
10 215 were considered observed (Iobs > 2σ(I)). Final unit cell param-
eters were determined by least-squares fit of 9928 reflections covering
2
4
1
(
0.44 g, 88%): mp decomposes at 115 °C; H NMR (CDCl , 400 MHz)
3
δ 6.15 (1H, s, H-2), 4.70, 4.61 (each 1H, s, H-29), 4.25, 3.85 (each 1H, d,
J = 12 Hz, H-28), 3.38 (2H, bs, NH ), 2.45 (1H, ddd, J = 11, 6 Hz, H-
2
1
9), 2.07 (3H, s, OCOCH ), 1.69, 1.15, 1.09, 1.08, 1.06, 0.97 (each 3H,
the resolution range of 8.280 to 0.800 Å. Data were corrected for
3
13
28c
s, 6 × CH ), 1.0−2.0 (CH, CH ); C NMR (CDCl , 100 MHz) δ 201.2,
absorption effects using the multiscan method (SADABS).
The
3
2
3
1
4
2
71.6, 150.0, 135.8, 129.0, 110.0, 62.8, 53.6, 48.7, 47.6, 46.3, 45.6, 44.2,
3.0, 41.6, 38.0, 37.7, 34.5, 33.8, 29.7, 29.5, 27.9, 27.0, 25.2, 21.74, 21.1,
1.1, 20.4, 19.1, 19.0, 16.4, 14.7; HREIMS m/z 495.3721 (calcd for
calculated minimum and maximum transmission coefficients (based on
crystal size) are 0.9763 and 0.9947. Statistical analysis with the XPREP
2
8d
program in SHELXTL indicated the space group was P2 . Routine
1
C H NO , 495.3712).
Further purification using gravity SGC, eluting with hexanes/EtOAc
direct methods structure solution was performed with SHELXTL, which
produced the majority of non-hydrogen atom coordinates on the single
12 molecule in the asymmetric unit. Upon initial refinement with
SHELXTL, the solvent in the voids of the asymmetric unit became
discernible in the Fourier difference map. However, subsequent re-
finement steps were unable to resolve the electron density into chem-
ically reasonable solvent molecules. Thus, application of the solvent
32
49
3
(
(
8:2), caused decomposition of the enamine 11, and the enolic ketone
NMR and MS data) was obtained in some of the fractions. Attempts
at recrystallization resulted in decomposition of 11, and when stored for
longer periods NMR analysis indicated decomposition to complex
mixtures.
8
68
dx.doi.org/10.1021/np400947d | J. Nat. Prod. 2014, 77, 863−872

Journal of Natural Products
Article
28e
removal utility in PLATON SQUEEZE
to the integrated data
a white gelatinous precipitate was observed. A mixture of petroleum
produced the solvent-free data set that was used to finalize the
refinement. After full isotropic refinement was completed, the thermal
parameters for all non-hydrogen atoms were then allowed to refine
anisotropically. Subsequently, hydrogen atoms were added at idealized
positions, and bond distances with their isotropic thermal parameters
were fixed at 1.5 times the Uiso values of their bonding partners for the
methyl hydrogens and 1.2 times the Uiso values of their bonding partners
for all others. The model was allowed to refine with hydrogens con-
strained as atoms riding on their bonding partners. This resulted in a
ether/EtOAc/Et O (2:2:1, 50 mL) was added. The mixture was stirred
until clear. The organic fraction was extracted from the aqueous layer
2
and dried (MgSO ), filtered, and concentrated to a colorless glass
4
(0.1 g), which was found to be a 50:50 mixture of acetylated products
1
15/16: H NMR (CDCl 400 MHz) δ 5.60 (1H, dd, J = 10, 6 Hz,
3,
H-2, 15), 4.93 (1H, s, H-3 16), 4.70 (s, 2H, H-29 15/16), 4.60 (m, 2H,
H-29 15/16), 4.26 (2H, d, J = 11 Hz, H-28 15/16), 3.85 (1H, d, J = 11
Hz 15/16), 2.48−2.42 (3H, m, H-1e (16), H-19, 15/16), 2.22 (1H, dd,
J = 13, 6 Hz, H-1e 15), 2.18, 2.14 (each 3H, s, C-2/3-OCOCH , 15/16),
3
final standard residual R value of 0.0521 for observed data and 0.0552
2.08 (6H, s, C-28-OCOCH , 15/16), 2.3−1.0 (m, CH, CH ), 1.69, 1.68,
1
3
2
2
for all data. Goodness of fit on F was 1.041, and the weighted residual on
1.21, 1.13, 1.11, 1.10, 1.09, 1.04, 1.02, 0.96, 0.84, 0.83 (each 3H, s,
2
13
F , wR , was 0.1399 for observed data and 0.1421 for all data. The final
CH , 15/16) ppm; C NMR (CDCl , 100 MHz) δ 209.3, 204.5, 171.6
2
3
3
Fourier difference map showed minimal electron density, with the
largest difference peak and hole having values of 0.320 and −0.236
(171.5), 170.5, 170.1, 149.9 (149.8), 110.0 (109.9), 84.0 (C-3, 16), 71.8
(C-2, 15), 62.7 (62.6), 57.2, 55.4, 57.2, 50.1 (50.04), 48.64 (48.62),
48.60, 47.6, 46.3 (46.2), 46.0, 43.5 (43.3), 42.78 (42.74), 41.2, 40.9, 38.1,
37.4 (37.39), 34.5, 33.8, 33.7, 30.9, 29.6, 29.5, 29.46 (29.46), 28.8, 27.1,
26.9, 24.9 (24.87), 24.7, 21.0 (3C), 20.9, 20.7, 20.6, 19.1, 19.0, 18.97,
18.4, 17.3, 16.8, 16.5, 16.2, 15.7, 14.7, 14.6; HRMS (APCI+) m/z
−3
electron Å , respectively. Final bond distances and angles were all
within expected and acceptable limits for the 12 molecule.
2
-Hydroxy-3-oxo-28-O-acetylbetulin (13) and 2-Oxo-3-hydroxy-
2
8-O-acetylbetulin (14). To a mixture of 2α/β-bromo-3-oxo-28-O-
+
acetylbetulin (7/8 1.0 g, 1.78 mmol) in 83% aqueous acetone (249 mL)
was added a solution of K CO (0.25 g, 1.81 mmol, ∼1 equiv) in H O
541.3898 [M + H] (calcd for C H O 541.3893).
34
53
5
2
3
2
Condensation of the Enamine 11 and the Hydroxy Ketones 13
and 14 (ref 20a). To a two-necked round-bottom flask fitted with a
dropping funnel and a reflux condenser was added enamine 11 (0.33 g,
20b
(
51 mL). The reaction mixture was heated under reflux for 24 h. The
initially cloudy mixture became clear at high temperatures, and TLC
(
DCM/acetone 2%) showed all starting material had reacted after 24 h.
0.66 mmol) followed by NH OAc (0.2 g, 2.6 mmol, 4 equiv) and MeOH
4
The cooled solution was concentrated to half volume, and a 1% aqueous
HCL solution was added until the pH was neutral (52 mL). The mixture
was extracted with Et O (3 × 80 mL), washed with 5% NaHCO (2 ×
(20 mL). The mixture was heated at reflux for 1 h to ensure dissolution,
and the 2/3-hydroxy ketone 13/14 mixture (0.33 g, 0.66 mmol) in
MeOH (0.5 mL) was added dropwise to the reaction mixture. The
2
3
5
0 mL) and H O (1 × 50 mL), dried (Na SO ), concentrated, and
solution was heated at reflux for 48 h under N and monitored by TLC.
2
2
4
2
redried under vacuum (1.0 g). NMR analysis of the crude sample
showed the major product as 2-α-hydroxy-3-oxo-28-O-acetylbetulin
compound 13, with 14 as a minor component. However once
chromatography was completed, the NMR indicated a 50:50 mixture
of the alcohols 13/14. The crude product was therefore taken forward in
the reaction with the enamino ketone to miminize the number of
potential products formed.
SGC: column size (23 × 4 cm); eluent DCM/acetone 1.5%; collected
at 20 mL × 1.5 min. Material eluted in fractions 28−42 was concentrated
to a colorless solid of ∼1 g. Analysis by NMR indicated a 50:50 mix-
ture of 13/14. Further attempts at purification of this mixture by
chromatography on silica gel eluting with hexanes/EtOAc (4:1) gave a
compound that was slow to elute and was found to be the 2-oxo-3-
hydroxy-28-O-acetylbetulin derivative 14, as the only compound
isolated. NMR assignments were made by comparing the data with
the crude material isolated initially, which was enriched with the
The reaction mixture was cooled, quenched with H O, and extracted
2
with DCM. The organic layer was washed with brine, followed by H O,
2
and dried (MgSO ). Concentration led to a yellow solid mixture (0.41g,
4
∼60% yield) separated by flash SGC gradient elution (hexanes/EtOAc,
7:3 → 5:5, column size 25 × 4 cm). Examination of the fractions by
NMR and MS showed the first fraction contained a dimer with a
molecular ion at m/z 974 (0.059 g), which was not purified further, the
second was enamine 11 (0.10 g), and a third fraction was concentrated
to a colorless solid, imidazole derivative 17: 0.08 g; mp 185−190 °C;
2
0
20
R = 0.24 (hexanes/EtOAc, 1:1); [α] +24 (c 0.1, CHCl ); [α] +33
f
D
3
D
1
(c 0.34, CH Cl); H NMR (CDCl 400 MHz) δ 4.67, 4.58 (each 1 H, s,
3
3,
H-29), 4.22, 3.82 (1H, d, J = 11 Hz, H-28), 2.96 (1H, d, J = 16 Hz, H-1e),
2.42 (1H, ddd, J = 11, 6 Hz, H-19), 2.07 (s, 3H, OCOCH ), 1.99 (1H, d,
3
J = 16 Hz, H-1a), 1.9−1.0 (CH, CH ), 1.66, 1.39, 1.36, 1.24, 1.19, 1.05,
2
13
0.97, 0.77 (each 3H, s, CH ); C NMR (CDCl , 100 MHz) δ 173.5
3
3
(C-3), 171.6 (C-31, OCOCH ), 164.8 (C-2), 150.0 (C-20),
3
2
-hydroxy-3-ketone and the pure 2-oxo-3-hydroxy derivative.
110.1(C-29) 102.0 (C-33, (CH ) C), 62.8 (C-28), 53.3 (C-5), 48.7 (C-
3
2
1
2
-α-Hydroxy-3-oxo-28-O-acetylbetulin (13): H NMR (CDCl3,
18), 48.4 (C-9), 47.7 (C-19), 46.4 (C-17), 42.83 (C-14), 42.81 (C-1), 40.9
(C-8), 38.7 (C-10), 37.7 (C-13), 36.3 (C-4), 34.6 (C-22), 33.1 (C-7), 30.7
(C-24), 29.7 (C-21), 29.68 (C-16), 27.2 (C-15), 25.3 (C-12), 24.6 (C-23),
24.1 (C-35, CH C), 23.8 (C-34, CH C), 21.4 (C-11), 21.1 (OCOCH ),
4
00 MHz) δ 4.68, 4.59 (each 1H, s, H-29), 4.52 (1H, ddd, J = 13, 6, 4 Hz,
H-2), 4.23 (1H, m, H-28), 3.84 (m, 1H, H-28), 3.55 (1H, d, J = 4 Hz,
OH-2, D O exchange experiment), 2.39−2.46 (m, 2H, H-19, H-1e),
2
3
3
3
2
1
2
4
2
.07 (s, 3H, OCOCH ,), 2.0−0.9 (CH, CH ), 1.66 (s, 3H, H-30) 1.16,
19.8 (C-6), 19.3 (C-30), 16.5 (C-25), 15.6 (C-26), 14.7 (C-27); HRMS
3
2
13
+
.13, 1.09, 1.08, 0.94 (each 3H, s, CH ); C NMR (CDCl , 100 MHz)
(APCI+) m/z 535.4262 [M + H] (calcd for C H N O , 535.4264).
3
3
35 55
2
2
16.7, 171.7, 149.9, 110.1, 69.6, 62.7, 57.8, 53.9 (2C), 50.0, 49.9, 48.7,
7.7, 46.3, 42.8, 42.4, 41.0, 37.9, 37.5, 34.0, 31.7, 27.0, 24.9, 24.5, 21.3,
Deacetylation of Compound 17. To a solution of 17 (0.07g,
0.13 mmol) in aqueous MeOH 88% (30 mL) and DCM (3 mL) was
added K CO (0.056 g, 4.05 mmol), and the mixture stirred for 2 days at
1.1, 20.99, 19.1, 16.6, 16.2, 14.6.
2
3
1
2
-Oxo-3-hydroxy-28-O-acetylbetulin (14): H NMR (CDCl 400
rt and monitored by TLC (DCM/MeOH 2%, starting material R = 0.38
3,
f
MHz) δ 4.68, 4.59 (each 1H, s, H-29), 4.23 (1H, d, J = 11 Hz, H-28),
.84 (3H, m, H-28, H-3), 3.42 (1H, d, J = 4.8 Hz, OH-3, D O exchange
experiment), 2.50 (d, 1H, J = 12.4 Hz, H-1e), 2.39−2.46 (1H, m, H-19),
product, R = 0.28). After 48 h, H O/DCM, 50:50 (40 mL), was added,
f
2
3
and the organic layer was separated, dried (MgSO ), and concentrated
2
4
in vacuo. The resulting yellow glass (36 mg) was purified by flash
column chromatography on silica gel to provide 17a as a colorless solid,
which was recrystallized from MeOH (16 mg, 44% yield): mp 288 °C;
[α]20 +35 (c 0.2, CHCl ); H NMR (CDCl , 400 MHz) δ 4.70, 4.61
2
0
1
4
2
.07 (s, 3H, OAc), 2.0−0.9 (CH, CH ), 1.67 (s, 3H, H-30), 1.25, 1.12,
2
13
.96, 0.79, 0.65 (each 3H, s, CH ); C NMR (CDCl 100 MHz) 211.4,
3
3,
1
71.7, 149.9, 110.0, 83.0, 62.7, 54.5, 53.5, 51.0, 50.3, 48.6, 47.6, 46.3,
5.5, 43.9, 42.8, 41.3, 37.4, 34.5, 34.0, 29.6, 29.5, 29.2, 27.0, 24.9, 21.0,
D
3
3
(each 1H, s, H-29), 3.80, 3.35 (each 1H, d, J = 8 Hz, H-28), 3.0 (1H, d,
J = 16 Hz, H-1e), 2.39 (m, H-19), 1.99 (1H, d, J = 16 Hz, H-1a), 2.01−
0.9, 18.4, 16.9, 16.3, 15.6, 14.7; HRMS (APCI+) m/z 499.3751 (M +
+
H) (calcd for C H O , 499.3787).
1.00 (CH, CH ), 1.69, 1.42, 1.39, 1.28, 1.23, 1.07, 1.01, 0.77 (each 3H, s,
32
51
4
2
+
The mixture of alcohols was acetylated for further characterization.
-O-Acetyl/3-O-acetyl-3/2-oxo- (15/16). To a solution of the
alcohols 13/14 (0.21 g, 0.42 mmol) in pyridine was added Ac O
CH ); HRMS (APCI+) m/z 493.4107 [M + H] (calcd for C H N O,
3
33 53
2
2
493.4158).
2
9
2-Bromoallobetulone 19. The solid acid montmorillonite clay
2
(
2 mL), and the colorless solution stirred at rt overnight under a drying
K10 was used to carry out the rearrangement of betulin to allobetulin
3
0
tube. TLC (hexanes/EtOAc, 4:1) showed complete conversion to
product, which was one spot by TLC. Iced H O (50 mL) was added, and
according to a procedure reported previously: mp 271 °C (colorless
30
crystals from DCM/MeOH 95%); [α]20 +40 (c 0.2, CHCl ) (lit. mp
2
D
3
8
69
dx.doi.org/10.1021/np400947d | J. Nat. Prod. 2014, 77, 863−872

Journal of Natural Products
Article
2
0
20
31,33
2
66−268 °C); [α] +65 (c 0.3, CHCl ). Allobetulin was oxidized with
161−165 °C, [α] +28 (c 0.4) according to the literature methods.
D
3
D
pyridinium dichromate to yield the keto derivative 18 (allobetulone)
To a solution of 24 (1 g, 2.14 mmol) in morpholine (20 mL) was added
sulfur (0.75 g, 23.4 mmol, 11 equiv). The reaction mixture was heated at
reflux for 3 h. The morpholine was removed by fractional distillation into
a collection flask immersed in a (dry ice/ispropyl alcohol) cooling bath.
The resulting dark brown residue was dissolved in DCM (80 mL)
and washed with aqueous 10% HCl (5 × 20 mL). The organic layer
20
27a
with mp 210−212 °C; [α] +68 (c 0.3, CHCl ) (lit. 229−231 °C;
D
3
22
[
α] +86 (c 1, CHCl ). A solution of the ketone 18 (0.5 g, 1.14 mmol)
D 3
in dry THF was cooled to 0 °C in an ice bath, and an ice cold solution of
PTAB (0.45 g, 0.119 mmol, 1.05 equiv) in THF (15 mL) was added in
one batch. The mixture became colorless in approximately 5 min, and
after 10 min stirring brine (30 mL) was added. The mixture was
was washed with H O (30 mL), and the mixture was stirred overnight
2
extracted with DCM (20 mL × 2) and washed with H O (20 mL × 2),
(∼20 h). The phases were separated, and the organic fraction was dried
2
and the organic phase was dried (MgSO ) and concentrated to a white
(MgSO ) and concentrated to a crude solid (2 g). Purification using
4
4
foam solid (0.566 g). Examination of the solid by 1D and 2D NMR
showed the major product in the mixture of epimers 19 was the 2α-
SGC (hexanes/Et O, 6:1) gave 25 as a foam (0.27 g, 25% yield,
2
20
crystallized from EtOH): mp 147−148 °C; [α] +15 (c 1.25, MeOH);
D
1
bromo ketone. Recrystallization from CH Cl/MeOH gave colorless
H NMR δ 4.71, 4.57 (each 1H, s, H-29), 3.63 (3H, s, −COOCH ), 2.95
3
3
2
0
needles of 2α-bromoallobetulone: mp 231−233 °C; [α] +49 (c 0.2,
(1H, ddd, J = 10.5, 4 Hz, H-19), 2.35 (1H, dd, J = 12, 2 Hz, H-1e), 2.24−
D
29
20
1
CHCl ) (lit. mp 234−235 °C, [α] +35 (c 3, CHCl ); H NMR data
2.15 (3H, m), 2.10 (1H, dd, J = 12, 2, Hz, H-3e), (CH, CH ), 1.65, 1.00,
3
D
3
2
13
for the crystalline mixture of 2α/β-bromo ketones (CDCl , 400 MHz) δ
0.97, 0.88, 0.83, 0.80 (each 3H, s, CH ); C NMR (CDCl , 400 MHz) δ
3
3
3
5
.15−5.06 (2H, m, H-2α/β), 3.72, 3.43 (each 2H, d, H-28), 3.51 (2H, s,
212.3, 176.6, 150.3, 109.7, 56.5, 56.45, 56.1, 55.7, 51.3, 50.2, 49.3, 46.9,
43.0, 42.5, 41.1, 39.0, 38.1, 36.9, 33.8, 33.3, 32.1, 30.6, 29.7, 25.3, 23.1,
21.0, 19.4, 18.9, 17.2, 15.6, 14.7; HRMS (APCI+) m/z 469.3678 (M +
H-19), 2.66 (1H, dd, J = 13, 6 Hz, H-1e (2α-Br)), 2.46 (1H, m, H-1e (2β-
Br)), 2.06 (1H, m, H-1a, (2β-Br)), 1.7 (1H, t, J = 13 Hz, H-1a (2α-Br)),
+
1
6
.7−0.7 (CH, CH ), 1.18, 1.12, 1.07, 1.00, 0.92, 0.89, 0.78 (each
H) (calcd for C H O , 469.3682).
2
31 48
3
H, CH ).
X-ray Crystal Structure of 25. a. Data Collection. A clear, colorless,
plate-like crystal (0.07 mm × 0.22 mm × 0.34 mm) of 25 grown from
a MeOH solution was mounted on the end of a thin glass fiber using
Apiezon type N grease and optically centered. Cell parameter mea-
surements and data collection were performed at 123 ± 1 K with a
Bruker APEX2 CCD diffractometer using Mo Kα (0.71073 Å) radiation.
A sphere of reciprocal space was collected using the Multi-Run scheme
3
2
-Azido-3-oxoallobetulone 20 (ref 21). Sodium azide (0.375 g, 5.85
mmol, 6 equiv) was added to a solution of 2-bromo-3-oxoallobetulone
19) (0.5 g, 0.96 mmol) in NMP (10 mL) and Ac O (0.5 mL). After
(
2
stirring at rt under N for 22 h, the reaction was quenched by the
2
addition of iced H O, and the resulting yellow precipitate was collected
2
by filtration and dried under vacuum. Column chromatography (SGC)
1
28a
afforded 20 (150 mg, 54%); H NMR (CDCl , 400 MHz) δ 4.30−4.22
available in the Bruker APEX2 data collection package. Three sets of
3
(
2H, m, H-2α, H-2β), 3.75, 3.43 (each 2H, d, H-28 α/β), 3.51 (s, 2H, H-
frames were obtained, with each set using 0.50°/frame steps to cover
182° in ω at fixed φ values of 0°, 120°, and 240°, respectively, for a total
of 1092 frames. Data were integrated and reduced using Bruker
1
9 α/β), 2.34 (1H, dd, H-1), 2.17 (1H, t, J = 12 Hz, H-1), 1.7−0.8
(
0
CH ), 1.14, 1.13, 1.12, 1.09 1.07, 1.01, 0.95, 0.93, 0.92 (2 × CH ), 0.89,
2
3
13
28b
.79, 0.78, 0.77 (each 3H, α/β CH ); C NMR corresponded with
SAINT, resulting in 99.9% coverage of all unique reflections to a
3
21
+
reported data; HRMS (APCI+) m/z 482.3755 (M + H) (calcd for
resolution of 0.830 Å with an average redundancy of 4.485.
C H N O , 482.3747).
b. Crystal Data. C H O , fw = 468.69, orthorhombic, P2 2 2 , a =
30
48
3
2
31 48
3
1 1 1
3
Reaction of 20 with PPh /H O. To a stirred solution of the azide 20
9.3498(8) Å, b = 15.3827(14) Å, c = 18.6860(17) Å, V = 2687.5(4) Å ,
3
2
−3
−1
(
0.150 g, 0.31 mmol) in dry THF (4 mL) was added a THF (1 mL)
Z = 4, ρ = 1.158 Mg m , μ(Mo Kα) = 0.072 mm , λ = 0.71073 Å,
c
solution of PPh (0.175 g, 0.66 mmol). The light yellow solution was
F(000) = 1032.
3
stirred for 17 h, with no evolution of N observed. TLC (successive
development used, hexanes 100% followed by hexanes/EtOAc, 9:1)
showed all the azide had reacted and the presence of more polar
c. Structure Solution and Refinement. A total of 22 151 reflections
were measured, of which 4939 were independent (Rint = 0.0501) and
4239 were considered observed (Iobs > 2σ(I)). Final unit cell parameters
were determined by least-squares fit of 5455 reflections covering the
resolution range of 9.343 to 0.897 Å. Data were corrected for absorption
2
products. H O (0.125 mL) was added, and the reaction was stirred for a
2
further 24 h. The reaction mixture was concentrated, and the crude
yellow solid was purified using column chromatography (gradient
elution; hexanes 100% → hexanes/EtOAc, 8:2) to afford the 2,3-
2
8c
effects using the multiscan method (SADABS).
The calculated
minimum and maximum transmission coefficients (based on crystal
1
diketone 21 (36 mg, 25%) [R = 0.58 (PhCH /EtOAc 20%); H NMR
size) are 0.9953 and 0.9756. Statistical analysis with the XPREP program
f
3
27a
28d
data for 21 were in agreement with reported data] and the enamine
in SHELXTL indicated the space group was P2 2 2 . Routine direct
1
1 1
2
1
6
2 (34 mg, 24%), which was recrystallized from DCM and MeOH: mp
methods structure solution was performed with SHELXTL, which
produced the majority of non-hydrogen atom coordinates on the single
molecule of 25 in the asymmetric unit. The remaining non-hydrogen
atoms were found in subsequent difference maps. After full isotropic
refinement was completed, the thermal parameters for all non-hydrogen
atoms were then allowed to refine anisotropically. Hydrogen atoms were
added at idealized positions, and bond distances with their isotropic
thermal parameters were fixed at 1.5 times the Uiso values of their
bonding partners for the methyl hydrogens and 1.2 times the Uiso values
of their bonding partners for all others. The model was then allowed to
refine with hydrogens constrained as atoms riding on their bonding
20
1
53 °C; [α] +21 (c 0.11, CHCl ); H NMR (CHCl , 400 MHz) δ
D
3
3
.19 (1H, s, H-1), 3.75 (1H, d, J = 8 Hz, H-28), 3.51 (1H, s, H-19), 3.43
(
1H, d, J = 8 Hz, H-28), 1.72−1.0.7 (ring CH ), 1.14, 1.08, 1.06, 1.00,
2
0
.91, 0.89, 0.78 (each 3H, s, 7 × CH ).; HRMS (APCI+) m/z 454.3690
3
+
(
M + H) (calcd for C H NO 454.3685).
30 48 2
Conversion of 20 into Dimer 23. 2-Azido-3-oxoallobetulone (20)
0.05 g, 0.104 mmol) was dissolved in dry THF (2 mL), and PPh3
0.05 g, 0.19 mmol, 1.8 equiv) was added. The reaction was stirred at rt
(
(
for 1 h before H O (0.05 mL) was added, and stirring was continued
2
overnight. The mixture was concentrated and dried under high vacuum.
EtOH (2 mL) and pTsOH (3.5 mg) were added, and the reaction was
stirred at rt for 6 days. The precipitate was filtered and dried to yield
dimer 23 as a yellow solid in 7% yield. Compound 23: mp chars
partners. This resulted in a final standard residual R value of 0.0526 for
1
2
observed data and 0.0631 for all data. Goodness of fit on F was 1.055,
2
and the weighted residual on F , wR , was 0.1338 for observed data and
2
20
1
>
300 °C; [α] +47 (c 0.1, CHCl ); H NMR (CDCl , 400 MHz) δ
0.1400 for all data. The final Fourier difference map showed minimal
D
3
3
7.13 (1H, s), 6.47 (1H, s), 3.76 (2H, d, J = 8.8 Hz, H-28), 3.48 (4H, m,
electron density, with the largest difference peak and hole having values
−
3
H-19, H-28), 1.8−0.78 (CH,CH ), 1.16, 1.12, 1.10, 1.04, 0.94 (2 ×
CH ), 0.82 (s, CH ); HSMS (APCI+) m/z 890.7042 (M + H) (100%
of base) (calcd for C H NO 890.7026).
of 0.328 and −0.201 electron Å , respectively. Final bond distances and
angles were all within expected and acceptable limits for the 25
molecule.
2
+
3
3
60
92
4
Methyl 2-Oxo-28-betulonate 25. Betulonic acid was prepared from
Methyl 2-morpholino-3-oxo-28-betulonate 26. When morpholine
was not removed by distillation and the reaction was stopped with the
addition of water followed by extraction with DCM (20 mL), the
reaction products differed. The organic layer was washed with water
betulin (1) and had the following physical properties: mp 235−239 °C
31
32
(
2
MeOH); [α]20 +25 (c 0.4, MeOH) (lit. mp 247−249 °C, lit. mp
D
20
50−254 °C, [α]
+32 (c 0.4)). Methylation afforded methyl
D
32
20
D
betulonate (24): mp 140−142 °C, [α] +21 (c 0.5, MeOH) (lit. mp
(10 mL), 10% HCl (10 mL), and saturated aqueous NaHCO (10 mL),
3
8
70
dx.doi.org/10.1021/np400947d | J. Nat. Prod. 2014, 77, 863−872

Journal of Natural Products
Article
dried, and concentrated to a crude solid. The reaction mixture was
Company LLC, Guilford, Maine, for providing scale-up amounts
of state of Maine birch bark.
separated using SGC to give, along with 25, the morpholino derivative
2
0
1
2
(
6 as a colorless solid: mp 125 °C; [α] +76 (c 0.5, CHCl ); H NMR
D 3
400 MHz, CDCl ) δ 5.96 (1H, s, H-1), 4.74, 4.61 (each 1H, s, H-29),
3
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AUTHOR INFORMATION
Corresponding Author
Tel: 480-965-3351. Fax: 480-965-2747. E-mail: bpettit@asu.edu.
Notes
The authors declare no competing financial interest.
In memoriam of Lee Williams, deceased September 3, 2013.
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ACKNOWLEDGMENTS
We acknowledge the financial support from grants R01CA90441-
1-05, 2R56CA090441-06A1, and 5-RO1CA90441-07-08 from
the Division of Cancer Treatment and Diagnosis, NCI, DHHS;
the Arizona Disease Control Research Commission; J. W.
Kieckhefer Foundation; Margaret T. Morris Foundation; the
Robert B. Dalton Endowment Fund; as well as Dr. William Crisp
and Mrs. Anita Crisp. In addition we are very pleased to thank
Prof. R. Dunlap and Mr. B. Deane as well as Hardwood Products
́
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